SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes

Nordlund, Gustav; Brzezinski, Peter; Ballmoos, Christoph von

doi:10.1038/ncomms5303

Cited by 34 publications

(41 citation statements)

References 36 publications

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“…Cytochrome bo 3 expression and purification . The quinol oxidase from E. coli was expressed from the plasmid pETcyo in the C43 strain and purified as reported in, see also.…”

Section: Methodsmentioning

confidence: 99%

Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

Berg

Block

Höök

et al. 2017

Israel Journal of Chemistry

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Respiratory oxidases are transmembrane enzymes that catalyze the reduction of dioxygen to water in the final step of aerobic respiration. This process is linked to proton pumping across the membrane. Here, we developed a method to study the catalytic turnover of the quinol oxidase, cytochrome bo3 from E. coli at single‐molecule level. Liposomes with reconstituted cytochrome bo3 were loaded with a pH‐sensitive dye and changes in the dye fluorescence, associated with proton transfer and pumping, were monitored as a function of time. The single‐molecule approach allowed us to determine the orientation of cytochrome bo3 in the membrane; in ∼70 % of the protein‐containing liposomes protons were released to the outside. Upon addition of substrate we observed the buildup of a ΔpH (in the presence of the K+ ionophore valinomycin), which was stable over at least ∼800 s. No rapid changes in ΔpH (proton leaks) were observed during steady state proton pumping, which indicates that the free energy stored in the electrochemical gradient in E. coli is not dissipated or regulated through stochastic transmembrane proton leaks, as suggested from an earlier study (Li et al. J. Am. Chem. Soc. (2015) 137, 16055–16063).

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“…Cytochrome bo 3 expression and purification . The quinol oxidase from E. coli was expressed from the plasmid pETcyo in the C43 strain and purified as reported in, see also.…”

Section: Methodsmentioning

confidence: 99%

Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

Berg

Block

Höök

et al. 2017

Israel Journal of Chemistry

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“…[289,297] Fusion of cellular membranes can also be regulated by cellular fusion proteins and viral fusogenic proteins. [290,[298][299][300][301] The most employed are soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (Figure 13c,d). [290,300,301] SNAREs can be divided into v-SNAREs that are incorporated into transport vesicles and t-SNAREs that are relevant to terminal/target vesicles.…”

Section: Fusionmentioning

confidence: 99%

“…[290,[298][299][300][301] The most employed are soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (Figure 13c,d). [290,300,301] SNAREs can be divided into v-SNAREs that are incorporated into transport vesicles and t-SNAREs that are relevant to terminal/target vesicles. Studies have shown that t-SNAREs form stable complexes that can bind with v-SNARE to form a stable helix bundle (Figure 13c).…”

Section: Fusionmentioning

confidence: 99%

Interface Engineering in Multiphase Systems toward Synthetic Cells and Organelles: From Soft Matter Fundamentals to Biomedical Applications

et al. 2020

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products of evolution over billions of years. They are featured by multiple hierarchical compartments performing specialized and exquisitely coordinated functions. The biological and genetic complexity of cells and subcellular components make understanding their physicochemical foundation piece by piece challenging. [1-5] Moreover, the mystery of how the very first cell, protocell, comes into exist in early earth scenario is unveiled yet. [6] Motivated by understanding protocell and biological cells, synthetic cells are being constructed. Started form the simplest form, aqueous droplet enclosed by a bilayer structure, researchers adopt a bottom-up approach to study machineries of biological cells, and explore possible forms of the protocell in early world conditions. [7] Synthetic cells are important to bridge the gap between cellular organism and nonliving matter. They refer to synthetic compartments that encapsulate a wide variety of molecules and mimic one or multiple cellular functions. By segregation of contents in different compartments, synthetic cells are now engineered to perform multiple processes and functions, including segregating genetic information, genetic circuits, protein synthesis, metabolism, growth, reproduce, recognition, intercellular crosstalk, and adaptivity to surroundings. [8-17] In these simplified cell models, cellular processes and signal pathways are reconstituted, providing insights for cell biology and offering new opportunities for designing smart cell-like carriers of therapeutics. [18-26] In addition, the hypothesis of "RNA world" has inspired the investigation of synthetic compartments as protocells, in which nucleotide permeation, compartmental division, the synthesis of macromolecular polynucleotide, and the polymerization of ribonucleic acids (RNA) are explored. [7,27,28] The beginning of synthesizing cell-like structures starts from amphiphiles self-assembled at liquid/liquid interfaces to form enclosed compartments. [29-32] Recently, techniques such as microfluidics facilitate the fabrication of complex compartments with high-order hierarchy with excellent control. [33-36] Formulations of compartmentalization can be diverse, there are reviews mainly focused on one particular type of synthetic compartments, such as lipid vesicles (liposomes), [22,30,37,38] polymer vesicles (polymersomes), [39-43] lipid-polymer hybrid Synthetic cells have a major role in gaining insight into the complex biological processes of living cells; they also give rise to a range of emerging applications from gene delivery to enzymatic nanoreactors. Living cells rely on compartmentalization to orchestrate reaction networks for specialized and coordinated functions. Principally, the compartmentalization has been an essential engineering theme in constructing cell-mimicking systems. Here, efforts to engineer liquid-liquid interfaces of multiphase systems into membrane-bounded and membraneless compartments, which include lipid vesicles, polymer vesicles, colloidosomes, hybrids, and coacervate droplets,...

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“…At this step, achieving a particular desired enzyme orientation, high membrane integration efficiency, as well as high overall activity is emphasized. Lipid membranes are further functionalized for fusion with either the addition of charged lipids or another form of a fusogen, for example, fusogenic soluble N‐ethylmaleimide‐sensitive factor activating protein receptor (SNARE) proteins . Building blocks formed in such a way can be fused together in a specific order to form functional devices comprising several building blocks.…”

Section: Challenges Recent Trends and New Ideas For Functional Devicesmentioning

confidence: 99%

“…Building blocks formed in such a way can be fused together in a specific order to form functional devices comprising several building blocks. In a recent example of system integration via membrane fusion, an intact bacterial respiratory chain, consisting of fumarate reductase, bo 3 quinol oxidase and the ATP synthase was assembled by SNARE‐mediated fusion . Within the same study, an isolated ATP synthase was, with the same method, integrated into Escherichia coli inverted membrane vesicles, which were lacking ATP synthase due to deletion.…”

Section: Challenges Recent Trends and New Ideas For Functional Devicesmentioning

confidence: 99%

Artificial Organelles for Energy Regeneration

et al. 2019

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cell's total energy budget. [2] The universal energy currency that is used for these purposes and can be found in all forms of life is adenosine triphosphate (ATP). A vast majority of the cellular energy demand is covered by either converting a large variety of energy-rich substrates into ATP in a process called oxidative phosphorylation, or by converting light (electromagnetic) energy into ATP in a process called photophosphorylation (or photosynthesis).These naturally existing energy conversions are valid in in the context of bottomup synthetic biology as well. In the latter, an artificial cell is envisioned as a compendium of functional modules, each hand-tailored to partially or entirely mimic one of the essential life processes, such as reproduction, growth, motility, etc. [3] Like their natural counterparts, all these reenvisioned synthetic processes are energy demanding, therefore, the deliberate design of synthetic cells should involve suitable energy management strategy, by, for example, continuous regeneration of ATP. Apart from the importance in the context of artificial cells, new energy conversion strategies can be considered as a standalone feature for enzymatic and cell-free biotechnology, wherein bottom-up synthetic biology might also deliver new and sustainable solutions.The notion of synthetic in terms of engineered and/or non-natural can be even expanded to other forms of energy (like electrical energy) and non-natural building blocks. The chemically driven ATP synthesis, as in oxidative phosphorylation, represents a spontaneous process, in which the electrons of a fuel (glucose, NADH) are transferred to an electron acceptor such as oxygen with the concomitant generation of proton gradient, which is afterwards stored again as chemical energy in ATP. In the realm of synthetic biology, other options might also be feasible, for example: can we use electrical energy and directly plug it in to drive biological processes [4,5] or make use of natural electron transfer mechanisms to produce electricity? [6] The Electron Transport Chain-a Natural Toolbox of Functional Parts for the Construction of Artificial OrganellesDuring the process of oxidative phosphorylation, electrons are passed from an electron donor ("fuel") with a more negative One of the critical steps in sustaining life-mimicking processes in synthetic cells is energy, i.e., adenosine triphosphate (ATP) regeneration. Previous studies have shown that the simple addition of ATP or ATP regeneration systems, which do not regenerate ATP directly from ADP and P i , have no or only limited success due to accumulation of ATP hydrolysis products. In general, ATP regeneration can be achieved by converting light or chemical energy into ATP, which may also involve redox transformations of cofactors. The present contribution provides an overview of the existing ATP regeneration strategies and the related nicotinamide adenine dinucleotide (NAD + ) redox cycling, with a focus on compartmentalized systems. Special attention is being paid to those approach...

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SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes

Cited by 34 publications

References 36 publications

Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

Interface Engineering in Multiphase Systems toward Synthetic Cells and Organelles: From Soft Matter Fundamentals to Biomedical Applications

Artificial Organelles for Energy Regeneration

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